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首页> 外文期刊>Cell cycle >Cdc5 blocks in vivo Rad53 activity, but not in situ activity (ISA).
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Cdc5 blocks in vivo Rad53 activity, but not in situ activity (ISA).

机译：Cdc5阻止体内Rad53活性，但不阻断原位活性（ISA）。

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摘要

DNA damage promotes the activation of a signal transduction cascade referred to as the DNA damage checkpoint. This pathway initiates with the Mec1/ATR kinase, which then phosphorylates the Rad53/Chk2 kinase. Mec1 phosphorylation of Rad53 is then thought to promote Rad53 autophosphorylation, ultimately leading to a fully active Rad53 molecule that can go on to phosphorylate substrates important for DNA damage resistance. In the absence of DNA repair, this checkpoint is eventually downregulated in a Cdc5-dependent process referred to as checkpoint adaptation. Recently, we showed that overexpression of Cdc5 leads to checkpoint inactivation and loss of the strong electrophoretic shift associated with Rad53 inactivation. Interestingly, this same overexpression did not strongly inhibit Rad53 autophosphorylation activity as measured by the in situ assay (ISA). The ISA involves incubating the re-natured Rad53 protein with gamma (3)(2)P labeled ATP after electrophoresis and western blotting. Using a newly identified Rad53 target, we show that despite strong ISA activity, Rad53 does not maintain phosphorylation of this substrate. We hypothesize that, during adaptation, Rad53 may be in a unique state in which it maintains some Mec1 phosphorylation, but does not have the auto-phosphorylations required for full activity towards exogenous substrates.
机译：DNA损伤促进了称为DNA损伤检查点的信号转导级联的激活。此途径始于Mec1 / ATR激酶，然后使Rad53 / Chk2激酶磷酸化。然后认为Rad53的Mec1磷酸化可促进Rad53自磷酸化，最终导致一个完全活跃的Rad53分子，该分子可以继续磷酸化对DNA损伤抗性重要的底物。在没有DNA修复的情况下，该检查点最终会在依赖于Cdc5的过程中被下调，此过程称为检查点适应。最近，我们表明Cdc5的过表达导致检查点失活以及与Rad53失活相关的强电泳位移的丧失。有趣的是，通过原位测定（ISA）测得，该相同的过表达并未强烈抑制Rad53自身磷酸化活性。 ISA涉及在电泳和蛋白质印迹后，将经过重新变性的Rad53蛋白与经γ（3）（2）P标记的ATP孵育。使用新近确定的Rad53靶标，我们显示，尽管ISA具有很强的ISA活性，但Rad53并不能维持该底物的磷酸化。我们假设，在适应过程中，Rad53可能处于独特的状态，在该状态下它可以维持一些Mec1磷酸化，但不具有针对外源底物的完整活性所需的自磷酸化作用。

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